Development and evaluation of a trehalose-contained solution formula to preserve hUC-MSCs at 4°C

Human mesenchymal stem cells (hMSCs) hold great promise in cell therapy and regenerative medicine. Various preclinical and clinical trials have been carried out to illustrate the therapeutic potential of these cells. However, one major challenge for manufacturing clinical grade hMSCs is the requisition of current good manufacturing practice (cGMP) grade practices in cell isolation, processing, storage, and distribution. Development of non-toxic and animal serum-free preservation medium is critical for storage and distribution of mesenchymal stem cells (MSCs). In this study, we developed a solution formula that could preserve MSCs at 4°C for up to 3 weeks. In the solution, trehalose is a key ingredient for maintaining survival of MSCs. Among the concentrations investigated, 40 mM trehalose showed the best outcome with the viability maintained more than 92.7 ± 1.5% for 7 days. Cells preserved in the solution formula for 3 weeks still remained about 70% viability, and produced results similar to those of freshly harvested hMSCs in terms of growth kinetics, expression profile of cell surface antigens, and differentiation potential. In summary, storage of MSCs in the medium makes it far easier for transporting the cells from processing units to clinical sites.     

In vitro selection of glyphosate-tolerant variants from long-term callus cultures of Zoysia matrella [L.] Merr

A complete protocol of in vitro selection and greenhouse screening for glyphosate-tolerant variants in manilagrass (Zoysia matrella [L.] Merr) was established in this study. Newly subcultured calli of more than 5?years?? old were transferred to selection medium containing 2?mM glyphosate. After two rounds of selection, 220 calli survived out of 840 and were transferred to regeneration medium without glyphosate. Regenerated plantlets were then transferred to regeneration medium containing 0.5?mM glyphosate to select tolerant plantlets. After 1-month growth, there were plantlets remained green and new shoots formed beside or on discolored explants. These surviving organisms were then transferred to fresh regeneration medium for further growth. Fully developed plantlets were transferred to a green house and then subjected to greenhouse screening by foliar spraying with 0.05?% glyphosate solution. Six glyphosate-tolerant plantlets, TP1-TP6, were obtained and proliferated for determination of sod-tolerance using morphological and physiological measurements. Fourteen days after foliar application with 0.1?% glyphosate, only TP5 showed enhanced sod-tolerance. The dark green color index value of TP5 was significantly higher than CK2, demonstrating that TP5 suffered less injury from glyphosate than CK2. Different physiological characters were also observed in CK1, CK2 and TP5. Significantly higher chlorophyll a content and catalase activity were observed in TP5 than in CK1. Fourteen days after treatment (DAT), the ion leakage, proline content and ascorbate peroxidase activity of CK2 and TP5 increased significantly, but the ion leakage of TP5 was significantly lower than that of CK2. The guaiacol peroxidase activity of TP5 increased significantly 14 DAT, and was significantly higher than that of CK1 and CK2. No change in shikimate content was observed in CK2 or TP5 14 DAT.     

Upregulation of Protein Phosphatase 2A and NR3A-Pleiotropic Effect of Simvastatin on Ischemic Stroke Rats

Ca²⁺ influxes are regulated by the functional state of N-methyl-D-aspartate receptors (NMDARs). Dephosphorylation of NMDARs subunits decreases Ca²⁺ influxes. NR3, a novel subunit of NMDARs, also decreases Ca²⁺ influxes by forming new NMDARs with NR1 and NR2. It is meaningful to uncover whether protein phosphatase 2A (PP2A) and NR3A play a role in the protective effect of Simvastatin on ischemic stroke. In the present study, the Sprague-Dawley rats were pretreated with Simvastatin for 7 days before middle cerebral artery occlusion was performed to mimic ischemic stroke. The results showed that Simvastatin decreased brain ischemic infarct area significantly while increasing the expression levels of PP2A and NR3A, thus dephosphorylating the serine sites of NR1 (ser896 and ser897) along with increased enzymatic activities of PP2A. The protein levels of NR3A decreased as the enzymatic activities of PP2A were inhibited by okadaic acid. The results indicated that Simvastatin could protect the cerebrum from ischemic injury through a signaling mechanism involving elevated levels of PP2A and NR3A, and that PP2A might involve in the regulatory mechanism of NR3A expression.     

小麦eIF3c基因片段的克隆及序列分析

根据真核翻译起始因子eIF3c的保守序列,搜索小麦EST,由拼接的序列设计引物,从普通小麦‘川麦107'幼苗总RNA中克隆出1102bp的小麦eIF3c1基因片段命名为WeIF3c1.序列分析表明,该片段推断的氨基酸序列与两个拟南芥、水稻、甜樱桃、人类、线虫、老鼠等物种的真核翻译起始因子eIF3c相比较同源性分别为69、61%、85%、72%、38%、36%和38%.     

拟南芥冷诱导CBF/DREB类结合因子的克隆及其生物信息学分析

CBF/DREB结合因子广泛存在于植物中,主要参与诸如干旱、高盐以及冷胁迫等相关基因的表达调控.以拟南芥DNA为模板,PCR扩增出3个冷诱导的CBF/DREB结合因子,即CBF1、CBF2及CBF3,利用生物信息学手段对三者的cDNA序列、氨基酸序列的相似性、组成成分、理化性质、疏水性/亲水性、功能域、进化树、二级结构及DNA保守域的三级结构等进行了较为全面的分析.结果显示,CBF1、CBF2和CBF3的氨基酸序列同源性很高,达到91.4%,且都属于亲水性蛋白,三者在理化性质以及蛋白质二级结构乃至三级结构上也极为相似.另外, CBF1和CBF2都不具跨膜结构,CBF3含有1个跨膜螺旋.     

Adult trees cause density‐dependent mortality in conspecific seedlings by regulating the frequency of pathogenic soil fungi

Negative density‐dependent seedling mortality has been widely detected in tropical, subtropical and temperate forests, with soil pathogens as a major driver. Here we investigated how host density affects the composition of soil pathogen communities and consequently influences the strength of plant‐soil feedbacks. In field censuses of six 1‐ha permanent plots, we found that survival was much lower for newly germinated seedlings that were surrounded by more conspecific adults. The relative abundance of pathogenic fungi in soil increased with increasing conspecific tree density for five of nine tree species; more soil pathogens accumulated around roots where adult tree density was higher, and this greater pathogen frequency was associated with lower seedling survival. Our findings show how tree density influences populations of soil pathogens, which creates plant‐soil feedbacks that contribute to community‐level and population‐level compensatory trends in seedling survival.     

单户与联户经营对高寒草甸土壤理化性质与植物多样性的影响

在甘南高寒草甸单户经营草场与联户经营草场进行野外群落学调查,分析其物种多样性和功能多样性的变化,以揭示不同经营方式下土壤理化性质对植物多样性的影响机制。结果显示：（1）联户草场内植物群落的Margalef指数、Simpson指数、Shannon-Winener指数较大,而Pielou指数在不同放牧经营方式下的差异并不显著。（2）不同经营方式下,土壤含水量、土壤全氮、土壤全磷和土壤有机碳质量分数表现为联户大于单户的趋势且差异显著（P<0.05）;单户草场土壤电导率与土壤pH大于联户草场;随着土层的加深土壤含水量、土壤全氮质量分数、土壤全磷质量分数、土壤有机碳质量分数和土壤电导率增加,而土壤pH在土层之间的差异并不显著。（3）联户经营方式下的功能丰富度、功能均匀度与功能离散度显著大于单户草场（P<0.05）。（4）相关性分析表明,植物群落物种多样性和功能多样性与土壤含水量、土壤全氮质量分数和土壤全磷质量分数存在正相关关系,土壤电导率和土壤pH与植物群落物种多样性和功能多样性存在负相关关系。冗余分析表明,联户经营方式下土壤理化性质对植物多样性的影响更显著,且0相似文献   

Correlation between the In Vitro Functionality of Stored Platelets and the Cytosolic Esterase-Induced Fluorescence Intensity with CMFDA

It has been hypothesized that the cytosolic esterase-induced fluorescence intensity (CEIFI) from carboxy dimethyl fluorescein diacetate (CMFDA) in platelets may related to platelet functions. In the present study, we measured the change of CEIFI in platelets during storage, and examined the correlations of CEIFI with the in vitro functionality of stored platelets, including the ADP-induced aggregation activity, hypotonic shock response, expression of CD62P as well as platelet apoptosis. The CEIFI of fresh platelets, when tested at 10 μM CMFDA, the mean fluorescence intensity index (MFI) was 305.9 ± 49.9 (N = 80). After 1-day storage, it was 203.8 ± 34.4, the CEIFI of the stored platelets started to decline significantly, and reduced to 112.7 ±27.7 after 7-day storage. The change in CEIFI is highly correlated to all four functional parameters measured, with the correlation coefficients being 0.9813, 0.9848, -0.9945 and -0.9847 for the ADP-induced aggregation activity, hypotonic shock response (HSR), expression of CD62P and platelet apoptosis respectively. The above results show that the CEIFI measurement of platelets represents well the viability and functional state of in vitro stored platelets. This may be used as a convenient new method for quality evaluation for stored platelets if this result can be further validated by the following clinical trials.     

Beneficial effects of inhaled NO on apoptotic pneumocytes in pulmonary thromboembolism model

Background

Lung ischemia–reperfusion injury (LIRI) may occur in the region of the affected lung after reperfusion therapy. Inhaled NO may be useful in treating acute and chronic pulmonary thromboembolism (PTE) due to the biological effect property of NO.

Methods

A PTE canine model was established through selectively embolizing blood clots to an intended right lower lobar pulmonary artery. PaO2/FiO2, the mPAP and PVR were investigated at the time points of 2, 4, 6 hours after inhaled NO. Masson’s trichrome stain, apoptotic pneumocytes and lung sample ultrastructure were also investigated among different groups.

Results

The PaO2/FiO2 in the Inhaled NO group increased significantly when compared with the Reperfusion group at time points of 4 and 6 hours after reperfusion, mPAP decreased significantly at point of 2 hours and the PVR decreased significantly at point of 6 hours after reperfusion. The amounts of apoptotic type II pneumocytes in the lower lobar lung have negative correlation trend with the arterial blood PaO2/FiO2 in Reperfusion group and Inhaled NO group. Inhaled nitric oxide given at 20 ppm for 6 hours can significantly alleviate the LIRI in the model.

Conclusions

Dramatic physiological improvements are seen during the therapeutic use of inhaled NO in pulmonary thromboembolism canine model. Inhaled NO may be useful in treating LIRI in acute or chronic PTE by alleviating apoptotic type II pneumocytes. This potential application warrants further investigation.

     

The Effect of Orexin-A on Cardiac Dysfunction Mediated by NADPH Oxidase-Derived Superoxide Anion in Ventrolateral Medulla

Hypocretin/orexin-producing neurons, located in the perifornical region of the lateral hypothalamus area (LHA) and projecting to the brain sites of rostral ventrolateral medulla (RVLM), involve in the increase of sympathetic activity, thereby regulating cardiovascular function. The current study was designed to test the hypothesis that the central orexin-A (OXA) could be involved in the cardiovascular dysfunction of acute myocardial infarction (AMI) by releasing NAD(P)H oxidase-derived superoxide anion (O₂ ⁻) generation in RVLM, AMI rat model established by ligating the left anterior descending (LAD) coronary artery to induce manifestation of cardiac dysfunction, monitored by the indicators as heart rate (HR), heart rate variability (HRV), mean arterial pressure (MAP) and left intraventricular pressure. The results showed that the expressions of OXA in LHA and orexin 1 receptor (OX₁R) increased in RVLM of AMI rats. The double immunofluorescent staining indicated that OX₁R positive cells and NAD(P)H oxidative subunit gp91phox or p47phox-immunoreactive (IR) cells were co-localized in RVLM. Microinjection of OXA into the cerebral ventricle significantly increased O₂ ⁻ production and mRNA expression of NAD(P)H oxidase subunits when compared with aCSF-treated ones. Exogenous OXA administration in RVLM produced pressor and tachycardiac effects. Furthermore, the antagonist of OX₁R and OX₂R (SB-408124 and TCS OX2 29, respectively) or apocynin (APO), an inhibitor of NAD(P)H oxidase, partly abolished those cardiovascular responses of OXA. HRV power spectral analysis showed that exogenous OXA led to decreased HF component of HRV and increased LF/HF ratio in comparison with aCSF, which suggested that OXA might be related to sympathovagal imbalance. As indicated by the results, OXA might participate in the central regulation of cardiovascular activities by disturbing the sympathovagal balance in AMI, which could be explained by the possibility that OXR and NAD(P)H-derived O₂ ⁻ in RVLM mediates OXA-induced cardiovascular responses.